Summary: Serological studies confirmed that influenza B viruses have undergone marked antigenic changes from the previous vaccine strain. The data were used by the USPHS and WHO to recommend inclusion of a new strain, a B/Hong Kong/330/01-like virus,in inactivated influenza vaccines for the 2002-2003 season. Additional strains (B/Shangdong/7/97 and B/Hong Kong/1434/02) that could be used for vaccine production were identified. Our laboratory produced new reference reagents for the B/Hong Kong/330/01, B/Hong Kong/1434/02 strains, and B/Shandgong/7/97 strain for use in standardizing vaccines to be used in the Northern Hemisphere during 2002-2003. The reference reagents facilitated qualification and release of approximately 95 million doses of influenza vaccine for the United States. In response to the appearance of avian influenza A virus subtypes in man and the continued circulation of H5 and H9 influenza A hemagglutinin subtypes in people and poultry in Hong Kong and China, we have developed preliminary reagents to be used for testing experimental vaccines produced against H5 and H9 influenza viruses. It is anticipated that additional reagents will be produced for other hemagglutinin subtypes identified from surveillance studies sponsored by NIH and CDC. Many wild type viruses with appropriate antigenic characteristics grow to relatively low titers in eggs. Influenza B viruses in particular grow poorly in eggs, and they have now become a limiting factor in vaccine production. Our laboratory has investigated methods to produce high growth influenza B viruses. In initial studies we have demonstrated that it is possible to transfer growth characteristics by reassorting influenza B viruses. Although genetic and amino acid sequences are highly conserved among influenza B viruses, we have used our reassortants to identify amino acids in several genes that may be involved in egg adaptation. Further understanding of growth charactersitics will be valuable in optimizing reassortants for vaccine uses. In collaboration with Dr. C.D. Atreya, we have also examined amino acid sequence motifs of the nucleoprotein gene of influenza A and identified a cleavage recognition motif for the pyrolysin branch of cellular subtilases. The role of this subtilase recognition site is currently unknown, but its high level of conservation suggests that it may be necessary for viral replication or play a role in disease manifestations. Further investigations are planned.